Project Summary/Abstract Individuals with human immunodeficiency virus type-1 (HIV) are 30x more likely to develop active tuberculosis (TB) than those without HIV. TB is the leading cause of death among HIV patients. Mycobacterium tuberculosis (M.tb), the causative agent of TB, is contained within granulomas, complex structures composed of macrophages, B and T cells, and a fibrous cuff, in the tissues of patients. These structures help limit M.tb growth, but also provide a niche for M.tb. Although increased HIV replication and altered granuloma structures have been reported in the lungs of HIV-TB co-infected patients, no work has focused on the interaction of M.tb and HIV specifically in granulomas and it is unclear how HIV alters M.tb granulomas. This is particularly important as active TB develops either from early progression of a primary granuloma or reactivation from an established latent granuloma. This proposal will investigate the effects of HIV infection on M.tb granulomas, using our group's previously established in vitro granuloma model, which shares many features with human TB granulomas, including cell aggregation, composition, and altered bacterial growth without clearance. The granulomas in this model are generated by adding M.tb to human peripheral blood mononuclear cells (PBMCs) collected from healthy latently TB infected (LTBI) or TB nave individuals. Our data suggest that addition of HIV-infected T cells to pre-formed in vitro M.tb granulomas generated with PBMCs from LTBI individuals reduces granuloma stability and increases M.tb growth; whereas HIV infection increases granuloma formation in PBMCs from TB nave individuals and M.tb increases HIV growth in these cells. These results indicate that the initial infection and resultant host immune status have distinct impacts on secondary infection. We hypothesize that HIV infection in pre-formed granulomas modeling latent TB leads to exacerbated cell death, and inhibition of macrophage and lymphocyte function. However, in TB nave individuals, HIV-driven immune activation promotes granuloma formation, which provides a niche for enhanced HIV replication and M.tb growth. This proposal will investigate the seemingly conflicting evidence for granuloma disaggregation and promotion by HIV, dependent on pre-existing M.tb infection and host immune status. The specific aims of this proposal are to: 1) Elucidate the effect of HIV infection on pre-formed M.tb granulomas and 2) Determine how pre-existing HIV infection affects subsequent M.tb-induced granulomas, and how this impacts HIV. For both models, granuloma structure and function will be characterized with biochemical, and microscopy and transcriptomic analysis of single cells. The broad, long-term objective of this research program is to investigate the molecular mechanisms of HIV-M.tb co-morbidity, with the ultimate goal of designing therapeutics that more effectively target HIV and M.tb, alone, and in co-infected individuals. Better understanding of the early impact that HIV has on M.tb granulomas will help elucidate the mechanisms of rapid exacerbation and death of the co-infected patients and design of better therapeutics active in granulomas.